Peptidic self-assembly for nucleic acid delivery

ABSTRACT

A polypeptide conjugate for use in a method for binding and/or internalization of the polypeptide conjugate to a mammalian cell having a transferrin receptor (TFRC) and/or receptor for advanced glycation end products (RAGE). The polypeptide conjugate may be used in a method for targeting of a drug delivery system or diagnostic delivery system.

This application is a continuation of U.S. patent application Ser. No.14/912,997, filed Feb. 19, 2016, now U.S. Pat. No. 9,981,047, issued May29, 2018, which is a 371 national phase of International Application No.PCT/EP2014/067651, filed Aug. 19, 2014, and claims priority to DenmarkApplication No. PA 2013 70453, filed Aug. 19, 2013, the disclosures ofwhich are incorporated, in their entirety, by this reference.

This invention relates to a polypeptide conjugate for use in a methodfor targeting of a drug delivery system or diagnostic delivery system,targeting at least two receptors.

BACKGROUND

One of the key challenges in medicine is to be able to targettherapeutic agents to the desired site of action. If site-specifictargeting of drugs can be achieved this will reduce the requiredtherapeutic dose to obtain a beneficial effect and may effectivelyreduce drug-induced toxicity and adverse effects. One way of achievingthese is to use a particulate drug carrier system for drug delivery andtargeting. Encapsulation or incorporation of drug molecules in certaindrug carriers (e.g., liposomes) can further attain protection againstdrug degradation or inactivation en-route to the target site.

The biological performance of particulate drug carriers is controlled bya complex array of physicochemical and physiopathological factors,depending on the route of administration. Generally, physicochemicalconsiderations include particle size distribution, shape,rigidity/deformability and surface characteristics (e.g., electriccharge, surface-bound polymers and their conformation, surface densityof targeting ligands). These factors, for instance, can not onlymodulate drug carrier circulation times in the blood, but also affecttheir tissue deposition patterns, mode of entry into cells andintracellular trafficking. Biological considerations that control drugcarrier performance include determinants of phagocytic/endocyticrecognition and ingestion, the ‘state-of-responsiveness’ of the hostdefense system, a wide range of anatomical, physiological andbiochemical barriers, and escape routes from vasculature. Theblood-brain barrier (BBB) is a formidable gatekeeper in the body, whichis formed at the level of the endothelial cells of the cerebralcapillaries and essentially composes the major interface between theblood and the brain. Indeed, BBB is the most important anatomical factorlimiting the development of new drugs and biologics for the centralnervous system. There have been numerous attempts to employ strategiesthat aid drug passage across the BBB. Among these, nanotechnology-basedapproaches have gained tremendous importance as some of them are capableof overcoming the limitations inherent to BBB passage, but theseapproaches are still in need of further optimization to increase theirefficacy. One of the most promising approaches for brain targeting issurface decoration of particulate carriers with ligands specific forcerberal capillary endothelial cells, which mediates internalizationand/or transcytosis of the bound carrier. In this respect, WO2011/005098discloses examples of targeting peptides with selectivity towards thehuman brain capillary endothelial cell line hCMEC/D3 such as the peptideGly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly (SEQ IDNO. 1) that can be grafted to particulate systems (e.g., liposomes).However, in a later publication van Rooy and colleagues (EuropeanJournal of Pharmaceutical Sciences 2012, 45, 330-335), demonstrated thatthe same peptide when coupled to liposomes did not significantlyincrease liposome uptake by the target brain capillary endothelialcells. Thus, the authors discontinued the project. This illustrates thedifficulty in the design and engineering of particulate carriers thatcan effectively target human brain capillary endothelial cells andpromote the internalization of therapeutic and diagnostic agents.

The present invention has chemically modified the same aforementionedpeptide SEQ ID NO. 1(Gly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly) describinga polypeptide conjugate (and its other forms thereof) that canefficiently target two receptors for binding and/or internalization.Accordingly, this invention provides a conjugate that can be used fortargeting of pharmaceutically acceptable substances such as drugs,diagnostic agents or delivery systems of drugs or diagnostic agents tocertain cell types.

SUMMARY

In one aspect, a polypeptide conjugate is disclosed which includes apolypeptide having a polypeptide sequence (SEQ ID NO. 1):Gly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly, or aderivative thereof which is at least 80% identical to polypeptidesequence (SEQ ID NO. 1); at least one moiety attached to thepolypeptide; and pharmaceutically acceptable salts or esters thereof.

In some embodiments, the at least one moiety is hydrophobic. In someembodiments, the conjugate is in an aggregate form of at least twomolecules of the polypeptide conjugate. In some embodiments, theaggregate is a particle of at least about 2 nm in diameter. In someembodiments, the aggregate includes a fiber form in any aspect ratio. Insome embodiments, the aggregate comprises a mixture of particles andfibres in free form as well as interconnected particles and fibres. Insome embodiments, wherein the at least one moiety is selected from: adrug molecule, a biological molecule, a surface-active agent, ahydrophobic molecule, a fluorescent molecule, and salts thereof. In someembodiments, the at least one moiety is a drug molecule and saltsthereof. In some embodiments, the at least one moiety is a biologicalmolecule. In some embodiments, the at least one moiety is asurface-active agent. In some embodiments, the at least one moiety is ahydrophobic molecule and salts thereof. In some embodiments, the atleast one moiety is a fluorescent molecule.

In some embodiments, the polypeptide of the conjugate comprises two ormore polypeptides of sequence (SEQ ID NO. 1), or derivatives thereofseparated by a spacer. In some embodiments, the polypeptide is attachedto the at least one moiety via a linker which is a chemical entity or acovalent bond. In some embodiments, the linker is a chemical entity. Insome embodiments, the linker is a covalent bond. In some embodiments,the linker is a sulfur-containing amino acid. In some embodiments, thelinker is cysteine or a cysteine derivative.

In some embodiments, the spacer is selected from: a chemical entity,covalent bond, or non-covalent bond. In some embodiments, the spacer isa chemical entity. In some embodiments, the spacer is covalent bond. Insome embodiments, the spacer is selected from: a chemical entity,covalent bond, or non-covalent bond a non-covalent bond

In some embodiments, the the spacer comprises an amino acid or aderivative thereof.

In some embodiments, the polypeptide conjugate is in an aggregatednano-particulate form wherein, the polypeptide conjugate is aggregatedinto a crystalline form.

In some embodiments, the polypeptide conjugate further comprising atleast one active principle attached to the polypeptide via one or morenon-covalent bonds. In some embodiments, the at least one activeprinciple is attached to the polypeptide via physical entrapment.

In some embodiments, the moiety is a liposome or viral capsule. In someembodiments, the moiety is a polymeric nanoparticle or a particulatesystem selected from inorganic and composite particles. In someembodiments, the moiety is in the form of inorganic particles. In someembodiments, the moiety is in the form of composite particles.

In some embodiments, the moiety comprises at least one active principleselected from an active pharmaceutical ingredient and a diagnosticagent. In some embodiments, the active principle is an activepharmaceutical ingredient. In some embodiments, the active principle isa diagnostic agent.

In some embodiments, the active principle is selected from an activepharmaceutical small molecule, a protein, a nucleic acid, an antisensemolecule, an expression conjugate that comprises a nucleic acid thatencodes a therapeutic protein of interest, a liposome, nanoparticles,diagnostic agents, markers of a disease of a central nervous systemdisorder, cancer, diabetes, antibodies, erythrocytes, erythrocyteghosts, spheroplasts, monoclonal antibodies, labeled monoclonalantibodies which bind a marker of a central nervous system disorder,cancer or diabetes, and/or a fragment of antibody or monoclonalantibody.

In some embodiments, the active principle is an active pharmaceuticalsmall molecule. In some embodiments, the active principle is a protein.In some embodiments, the active principle is a nucleic acid. In someembodiments, the active principle is an antisense molecule. In someembodiments, the active principle is an expression conjugate. In someembodiments, the expression conjugate is a nucleic acid that encodes atherapeutic protein of interest. In some embodiments, the activeprinciple is a liposome. In some embodiments, the active principle is ananoparticle or nanoparticles. In some embodiments, the active principleis a diagnostic agent. In some embodiments, the active principle is amarker of a disease of a central nervous system disorder. In someembodiments, the active principle is a marker of cancer. In someembodiments, the active principle is a marker of diabetes. In someembodiments, the active principle is an antibody. In some embodiments,the active principle is an erythrocyte, In some embodiments, the activeprinciple is an erythrocyte ghost. In some embodiments, the activeprinciple is a spheroplasts. In some embodiments, the active principleis a monoclonal antibody. In some embodiments, the active principle is alabeled monoclonal antibody. In some embodiments, the active principleis a fragment of an antibody. In some embodiments, the active principleis a fragment of a monoclonal antibody.

In some embodiments, the polypeptide is a derivative that is at least90% identical to polypeptide sequence (SEQ ID NO. 1). In someembodiments, the moiety is a photo sensitive molecule or particle. Insome embodiments, the moiety is attached via a photo sensitive linker tothe polypeptide. In some embodiments, the aggregate also includescysteine and at least one hydrophobic moiety.

In some embodiments, the aggregate has a spherical structure. In someembodiments, the aggregate has a rod-shaped structure. In someembodiments, the active principle is surrounded within the aggregate.

In some embodiments, the polypeptide includes the sequenceGly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly-Gly-Gly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly(SEQ ID NO. 2). In some embodiments, the immediately aforementionedsequence has an additional cystein residue. In some embodiments, theadditional cystein residue is at the C-terminus of the peptide. In someembodiments, the additional cystein residue is at the N-terminus of thepeptide. In some embodiments, the additional cystein residue is locatedsomewhere between two existing residues.

In one aspect, a pharmaceutical composition is disclosed, having apolypeptide conjugate as herein described and a pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutical compositionincludes one or more pharmaceutically acceptable excipients. In someembodiments, the pharmaceutical composition is in the form of a solid.In some embodiments, the pharmaceutical composition is in the form of aliquid.

In another aspect, a method for delivering a therapeutic agent isdisclosed which includes (a) providing a polypeptide conjugate and (b)contacting a mammalian cell with the polypeptide conjugate. In someembodiments, the method also includes irradiating the polypeptideconjugate.

In some embodiments, the mammalian cell is from a mammalian tissueselected from the gastro-intestinal tract, bone marrow, liver, spleen,brain, kidney, lungs, pancreas, bladder, eye, normal and pathologicblood vessels, and cancer cells. In some embodiments, the mammalian cellis from the gastro-intestinal tract. In some embodiments, the mammaliancell is from the bone marrow. In some embodiments, the mammalian cell isfrom the, liver. In some embodiments, the mammalian cell is from thespleen. In some embodiments, the mammalian cell is from the brain. Insome embodiments, the mammalian cell is from the kidney. In someembodiments, the mammalian cell is from the lungs. In some embodiments,the mammalian cell is from the pancreas. In some embodiments, themammalian cell is from the bladder. In some embodiments, the mammaliancell is from the eye. In some embodiments, the mammalian cell is fromthe normal blood vessels. In some embodiments, the mammalian cell isfrom the pathologic blood vessels. In some embodiments, the mammaliancell is from cancer cells.

In another aspect, a use of a polypeptide conjugate or a compositionwith a polypeptide conjugate is disclosed for the treatment orprophylaxis of a disorder or diagnosis of a disorder. In someembodiments the use is for the treatment of a disorder. In someembodiments, the use is for the diagnosis of a disorder.

In some embodiments, the disorder is selected from a central nervoussystem disorders, cancer, and diabetes. In some embodiments, thedisorder is a central nervous system disorder. In some embodiments, thedisorder is cancer. In some embodiments, the disorder is diabetes.

In some embodiments, the central nervous system disorder is selectedfrom depression, dementia, prion diseases, Alzheimer's disease,Parkinson's disease, multiple sclerosis, amylotrophic lateral sclerosis,and schizophrenia. In some embodiments, the central nervous systemdisorder is depression. In some embodiments, the central nervous systemdisorder is dementia. In some embodiments, the central nervous systemdisorder is prion diseases. In some embodiments, the central nervoussystem disorder is Alzheimer's disease. In some embodiments, the centralnervous system disorder is Parkinson's disease. In some embodiments, thecentral nervous system disorder is multiple sclerosis. In someembodiments, the central nervous system disorder is amylotrophic lateralsclerosis. In some embodiments, the central nervous system disorder isschizophrenia.

In some embodiments, the disorder is traumatic brain injury. In someembodiments, the disorder is psychosis. In some embodiments, thedisorder is Chorea. In some embodiments, the disorder is Huntingtondisease. In some embodiments, the disorder is encephalopathy. In someembodiments, the disorder is epilepsy. In some embodiments, the disorderis a cerebrovascular disease. In some embodiments, the disorder is aneurodegenerative disorder.

In some embodiments, the disorder is lyme disease. In some embodiments,the disorder is poliomyelitis.

In some embodiments, the disorder is cancer. In some embodiments, thecancer is selected from carcinoma cancer, breast cancer, prostatecancer, lung cancer, pancreatic cancer, colon cancer, sarcoma cancers,bone sarcoma, sarcoma of cartilage, sarcoma of fat tissues, nervecancer, lymphoma, leukemia, germ cell tumor, seminoma, dysgerminoma,blastoma cancer. In some embodiments, the cancer is carcinoma cancer. Insome embodiments, the cancer is breast cancer. In some embodiments, thecancer is prostate cancer. In some embodiments, the cancer is lungcancer. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is colon cancer. In some embodiments, the canceris sarcoma cancer. In some embodiments, the cancer is bone sarcoma. Insome embodiments, the cancer is sarcoma of cartilage. In someembodiments, the cancer is sarcoma of fat tissues. In some embodiments,the cancer is nerve cancer. In some embodiments, the cancer is lymphoma.In some embodiments, the cancer is leukemia. In some embodiments, thecancer is germ cell tumor. In some embodiments, the cancer is seminoma.In some embodiments, the cancer is dysgerminoma. In some embodiments,the cancer is blastoma cancer.

In some embodiments, the disorder is diabetes. In some embodiments, thedisorder is diabetes mellitus. In some embodiments, the disorder is type1 diabetes. In some embodiments, the disorder is type 2 diabetes. Insome embodiments, the disorder isgestational diabetes.

The present invention relates to a polypeptide conjugate for use in amethod for binding and/or internalization of the polypeptide conjugateto a mammalian cell having a transferrin receptor (TFRC) and/or receptorfor advanced glycation end products (RAGE), the method comprising thesteps of

a. providing

i. a polypeptide conjugate comprising a polypeptide attached to at leastone moiety, wherein the polypeptide sequence isGly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly (SEQ ID NO.1), or a derivative being at least 80% identical to polypeptide sequence(SEQ ID NO. 1), or pharmaceutically acceptable salts or esters thereofand

ii. a mammalian cell having a TFRC and/or RAGE,

b. allowing interaction of polypeptide conjugate with the mammalian cellhaving the TFRC and/or RAGE, and

c. binding of the conjugate to the TFRC and/or RAGE, and/orinternalization of the polypeptide conjugate or a component thereof intothe mammalian cell having the TFRC and/or RAGE.

The ability of polypeptide sequence (SEQ ID NO. 1) or derivativesthereof to interact with the TFRC and/or RAGE receptor makes it possibleto bind or internalize a conjugate comprising the polypeptide sequence(SEQ ID NO. 1) or derivatives thereof into a mammalian cell having theTFRC and/or RAGE receptor. The moiety associated with the polypeptidesequence (SEQ ID NO. 1) may be biological, chemical or a particulateentity, which may be used either for therapeutic or diagnostic purposesor both. In certain embodiments the moiety is released from thepolypeptide to exert a biological effect inside a cell. Alternatively,the moiety may be released outside the cell. Since TFRC and/or RAGEreceptors are present only on some cells this will make it possible totarget the moieties specifically towards cells that express thesereceptors.

The polypeptide conjugate provide means for use as a targeting principlein the treatment, prophylaxis and diagnosis of a disorder associatedwith the central nervous system and/or a malignant tissue bearing cellsexpressing TFRC and/or RAGE on their plasma membrane. The peptide partof the conjugate plays an important role in the interaction with theTFRC and/or RAGE through formation of salt bridges, electrostaticinteractions, hydrogen bonding, van der Waals and/or hydrophobicinteractions.

Since the RAGE receptors are expressed on cells lining the blood brainbarrier the peptide conjugate is able to cross this barrier. As inferredby real-time single cell imaging these cells take up the polypeptideconjugate (or its other forms thereof) through different modes ofinternalization processes within minutes to hours after initial bindingof the peptide conjugate to the corresponding receptors.

In order for the polypeptide to be able to bind to the TFRC and/or RAGE,preferably, the peptide comprise an amino acid sequence which is atleast 12, 13, 14 amino acids identical to the polypeptide correspondingto an 80%, 87% and 93% identity to the polypeptide sequence (SEQ ID NO.1). In an aspect of the invention the derivative is at least 90%identical to polypeptide sequence (SEQ ID NO. 1). The total number ofamino acids of the polypeptide may be higher than the amino acids ofsequence (SEQ ID NO. 1) or derivatives thereof, such as 16, 17 or 18amino acids.

In another aspect of the invention the polypeptide conjugate is in anaggregated form prior to interaction of the polypeptide conjugate withthe mammalian cell having the TFRC and/or RAGE, wherein the aggregate isformed of at least two molecules of the polypeptide conjugate. Theaggregated form is relatively stable to external conditions so that theconjugate will be less vulnerable to degradation.

Notably, the aggregate may be a particle of at least 2 nm in diameter.The particle may have any suitable physical form or shape, including afiber form in any aspect ratio. The particles may self-aggregate toconceal the moiety inside a core or surrounded by fibers. The concealingof the moiety inside a particle isolates it from non-target cells. Thus,cells not harbouring TFRC and/or RAGE receptors may remain unaffectedand less vulnerability to adverse effects of the moiety. The moieties ofthe conjugate may be considered the “core” of a particle, whereas thepolypeptide in any form may be considered the “shell”.

In one embodiment, the peptide conjugates forming core-shell structurednanoparticles with a hydrophobic core comprising the moiety and ahydrophilic peptide shell comprising amino acids from peptide (SEQ IDNO. 1) arranged towards the surroundings. This structure may furtherfold into a tertiary structure. In a particular embodiment, where themoiety or hydrophobic core is a fluorophore (e.g., FAM) the criticalaggregation concentration (CMC) is 2.8 μM. Above the CMC the peptideconjugate can self-assemble and form a network of particles and fibres,that is a mixture of particles and fibres in free form or interconnectedparticles and fibres. In this particular embodiment, the sizes ofparticles formed have a hydrodynamic diameter in the range of 70-170 nmat physiological pH as determined by nanoparticle tracking analysis(NTA).

It is contemplated that the at least one moiety of the peptide conjugatemay be any drug molecule, biological molecule, a surface-active agent, ahydrophobic molecule or fluorescent molecule or salts thereof. Theversatility of the technology makes it possible to design a variety ofconjugates which may be used for any medical or non-medical purpose. Inthe medical field, the invention provides a general method fortransporting moieties to cells harbouring TFRC and RAGE. Thus, accordingto an aspect of the invention, the polypeptide conjugate is provided inaggregated form in a pharmaceutical composition. The pharmaceuticalcomposition may be used for the treatment or prophylaxis of diseases.

Once the polypeptide conjugate has targeted the tissue of interest thepharmaceutical or diagnostic effect can take place due to the bindingand/or internalizing properties of the peptide conjugate or a drugmolecule carried by the peptide conjugate.

In one embodiment, the polypeptide or derivatives thereof may be coupledby a spacer to one or more polypeptides of the polypeptide (SEQ ID NO.1), or derivatives thereof. In a particular embodiment the polypeptide(SEQ ID NO. 1) is coupled to another polypeptide (SEQ ID NO. 1), thusforming a dimer formed of the polypeptides. Surprisingly, the presentinvention further shows a conjugate comprising a dimer of thepolypeptide has higher binding and affinity for cells expressing TRFCand/or RAGE. The more binding and/or higher affinity serves tofacilitate the transfer of the moiety and thus enhances the bindingand/or internalization.

The spacer may be a chemical entity, covalent bonding, or non-covalentbonding. In a specific example the spacer comprises an amino acidresidue such as a glycine or serine residue or a derivative thereof orany other chemical structures such as amide bonds.

In another embodiment the polypeptide is attached to at least one moietyvia a linker, which may be a chemical entity or a covalent bonding. Thepresence of the linker makes it possible in a convenient way to producea variety of different conjugates, which may be used for differentpurposes, such as different diseases.

The polypeptide (SEQ ID NO. 1) may be coupled directly to the moietywith a covalent bond or the linker may comprise a sulphur-containingamino acid residue such as cysteine or methionine, or a derivativethereof. When a sulphur-containing amino acid residue is used it makesit possible to couple two components to the same polypeptide. The twocomponents include at least one moiety as used herein, however bothcomponents may be moieties thereby doubling the effect. The othercomponent may be different active compounds, markers, fluorescentmolecules, etc. specifically, one of the components may be a liposome.

An active principle is defined as a constituent of a drug on which thecharacteristic therapeutic action of the substance largely depends. Theactive principle is selected from a group of active pharmaceutical smallmolecules, proteins, nucleic acids including siRNA molecules andantisense molecules, expression conjugates that comprise a nucleic acidthat encodes a therapeutic protein of interest, liposomes,nanoparticles, diagnostic agents, markers of a disease of centralnervous system disorder, cancer or diabetes, antibodies, erythrocytes,erythrocyte ghosts, spheroplasts, monoclonal antibodies, labeledmonoclonal antibodies which bind a marker of a central nervous systemdisorder, cancer or diabetes, and/or a fragment of an antibody or amonoclonal antibody. In a certain embodiment, the moiety of thepolypeptide conjugate is attached to the polypeptide via one or morenon-covalent bonding(s). The non-covalent bonding includes the physicalentrapment or encasement of an active principle. The moiety of thepolypeptide conjugate may be any conventional case such as a viralcapsule, liposome, etc. which has entrapped an active principle.

In one embodiment of the polypeptide conjugate the moiety is a liposome.The liposome is contemplated to comprise an active principle or adiagnostic agent, and transport it in circulation of the body to thetarget cell.

In another embodiment the moiety may be a polymeric nanoparticle, suchas nanoparticles prepared from polymers with the ability to dissolve,entrap, encapsulate or attach to an active principal, or any otherparticulate system including an inorganic and composite particle such asmetallic particles, for example gold, silver, cadmium, selenide orsilicon or combinations thereof.

In a preferred embodiment the polypeptide conjugate may target thegastro-intestinal tract, bone marrow, liver, spleen, brain, kidney,lungs, pancreas, bladder, eye, normal and pathologic blood vesselsand/or cancer cells.

It is contemplated that the polypeptide conjugate may be administered asa solid composition to a patient in need thereof for example in a tabletor capsule form. It is also contemplated that the polypeptide conjugatemay be administered as a solution/suspension composition to a patient inneed thereof.

In a particular embodiment the moiety is a photo sensitive molecule orparticle. Photosensitive molecules or particles attached to thepolypeptide conjugate are able to target cells and thus may be used asfunctional, therapeutic and/or diagnostic tools. Photosensitivemolecules may comprise fluorophores such as cyanine or derivativesthereof, preferably Cy3 or Cy5, fluorescein amidite (FAM), or rhodamine.

In a more particular embodiment the moiety is attached via aphotosensitive linker to the polypeptide allowing the liberation ofpolypeptide conjugate components upon exposure to irradiation. Thepolypeptide conjugate in the cell may be exposed to irradiation withwavelengths in the range of 400-800 nm for a specific time (e.g., up to10 min) activating the photosensitive bonding, thereby liberating themoiety. The chosen wavelength depends on the choice of the fluorophorand is within skill of the art. The photo sensitive linker may be adisulphide-bridge coupled to cyanine by a covalent bonding in aCy3-siRNA:FAM-polypeptide complex. When irradiated with light having awavelength of 488 nm the Cy3-siRNA dissociates from the FAM-labeledpolypeptide. Other photosensitive linkers may be biotin, 2-nitrobenzyl,phenacyl esters or the like.

In another aspect of the invention the polypeptide conjugate comprises apolypeptide linked through a linker to a hydrophobic moiety, wherein thepolypeptide sequence isGly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly (SEQ ID NO.1), or a derivative being at least 80% identical to polypeptide sequence(1), or pharmaceutically acceptable salts or esters thereof, inaggregated nano-particulate form wherein, the polypeptide conjugate isaggregated into a crystalline form.

In yet another aspect of the invention the polypeptide conjugatecomprises a polypeptide linked through a linker to a hydrophobic moiety,wherein the polypeptide sequence isGly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly (SEQ ID NO.1), or a derivative being at least 80% identical to polypeptide sequence(SEQ ID NO. 1), coupled by a spacer to one or more polypeptidesequence(s) (SEQ ID NO. 1) or derivative(s) thereof at least 80%identical to polypeptide sequence (SEQ ID NO. 1), or pharmaceuticallyacceptable salts or esters thereof.

In a further embodiment, the polypeptide conjugate may be used for thetreatment or prophylaxis of a disorder, wherein the disorder may beselected from but not limited to the groups consisting of centralnervous system disorder, cancer, diabetes, cardiovascular, inflammatoryor diagnosis as well as cosmetic conditions such as wrinkles, allergiesor any other skin problem. The polypeptide conjugate may be used as adiagnostic tool to diagnose central nervous system disorder, cancer, ordiabetes disorders or any other disorder.

The central nervous system disorder may be selected from the groupconsisting of depression, dementia, prion diseases, Alzheimer's disease,Parkinson's disease, multiple sclerosis, amylotrophic lateral sclerosis,schizophrenia, lyme disease, poliomyelitis, traumatic brain injury,psychosis, chorea, Huntington disease, encephalopathy, epilepsy,cerebrovascular diseases, neurodegenerative disorders and centralnervous system cancer, the cancer may be selected from carcinoma cancer,i.e. breast, prostate, lung, pancreas, and colon cancer, sarcoma cancer,i.e. bone, cartilage, fat and nerve cancer, lymphoma cancer andleukemia, germ cell tumor i.e. seminoma or dysgerminoma cancer, blastomacancer, the diabetes i.e. diabetes mellitus may be selected from type 1,type 2, gestational diabetes.

It is contemplated that the polypeptide conjugate may be used in apharmaceutical composition where the pharmaceutical compositioncomprises the peptide conjugate and one or more pharmaceuticallyacceptable excipients.

Definitions

As used herein, the term “polypeptide conjugate” is to be understood asa polypeptide of the invention which is linked to a moiety (the term“moiety” is to be understood as defined herein) and which bind to TFRCand/or RAGE in vitro and/or in vivo.

As used herein, the term “moiety” refers to one or two or more part(s)of a polypeptide conjugate (the term “polypeptide conjugate” is to beunderstood as defined herein) into which something may be separated fromthe polypeptide part of the polypeptide conjugate such as, but notlimited to, an amino acid, a nucleic acid, a liposome and/or photosensitive molecule.

As used herein, the term “transferrin receptor” (TFRC) refers to amembrane glycoprotein known to mediate cellular uptake of iron from aplasma glycoprotein, transferrin. Iron uptake from transferrin involvesthe binding of transferrin to the TFRC and internalization oftransferrin within an endocytic vesicle by receptor-mediatedendocytosis. The iron is released from the protein by a decrease inendosomal pH. With the exception of highly differentiated cells, TFRCsmay be expressed on all cells but their levels vary greatly. TFRCs arehighly expressed on immature erythroid cells, placental tissue, andrapidly dividing cells, both normal and malignant (Ponka P and Nam C:The transferrin receptor: role in health and disease: 1999,31:1111-1137). The term “transferrin receptor” may also refer to afusion protein of the transferring receptor.

As used herein, the term “receptor for advanced glycation end products”(RAGE) refers to a member of the immunoglobulin super-family, encoded inthe Class III region of the major histocompatability complex. Receptorfor advanced glycation end product is highly expressed only in the lungat readily measurable levels but increases quickly at sites ofinflammation, largely on inflammatory and epithelial cells. It is foundeither as a membrane-bound or soluble protein that is markedlyup-regulated by stress in the endothelium, smooth muscle cells, cardiacmyocytes, neural tissue (including CNS and brain), and mononuclear cellsthereby regulating their metabolism and enhancing their central barrierfunctionality (Louis J et al: RAGE (Receptor for Advanced Glycation Endproducts), RAGE Ligands, and their role in Cancer and Inflammation,Journal of Translational Medicine 2009, 7:17 and P. Alexiou et al: AMulti-Ligand Receptor Unveiling Novel Insights in Health and Disease,Current Medicinal Chemistry 2010, 17: 2232-2252) The term “receptor foradvanced glycation end products” may also refer to a fusion protein ofthe receptor for advanced glycation end products.

As used herein, the term “polypeptide” means a compound that consists ofamino acids that are linked by means of peptide bonds e.g. covalentamide linkage formed by loss of a molecule of water between the carboxylgroup of one amino acid and the amino group of a second amino acid.

As used herein, the term “nanoparticle” means a particle with a diameterbetween 0.1 and 1000 nm, e.g. liposomes, polymer micelles, polymer-DNAcomplexes, nanospheres, nanofibres. All these nanoparticles are known inthe art. The surface of such nanoparticles is often modified byPEGylation, i.e. polyethylene glycol (PEG) is attached to the surface ofthe nanoparticles.

As used herein, the term “identical” or “identity” means, that analignment of two sequences within a stretch of a defined number of aminoacids (in the present invention: 15 amino acids) comprises the indicatednumber of identical amino acids, i.e. the term “identical” or “identity”is as equal to the number of exact matches in an alignment of an aminoacid sequence of the present invention and a different amino acidsequence or length. An exact match occurs when the amino acid sequencehave identical amino acid residues in the same position overlap. In oneembodiment the identity can be expressed in percentage by dividing thenumber of exact matches by length of the shorter of the two amino acidssequences and convert the result into percentage.

As used herein, the term “mammalian cell” includes in vitro cells,including cultured cells, and/or in vivo cells from animals of economicimportance such as bovine, ovine, and porcine animals, especially thosethat produce meat, as well as domestic animals, sports animal, zooanimals and humans, the latter being preferred.

The term “pharmaceutical composition” encompasses a product comprisingan optional carrier comprising inert ingredients.

As used herein, the term “pharmaceutically acceptable” refers tophysiologically well tolerated by a mammal or human.

As used herein, the term “spacer” for example a peptide bond or an aminoacid, a linker peptide between at least two polypeptide sequences ofpolypeptide (SEQ ID NO. 1).

As used herein, the term “linker” mean a structure that links a peptideaccording to the invention and a pharmaceutically acceptable substance(the term pharmaceutically acceptable substance” is to be understood asdefined herein) by covalent or non-covalent bonds. The term “linking”includes, but is not limited to: a linker peptide, a carbohydrogen bond,streptavidin-biotin, polyethylene glycol (PEG), a disulfide bridge,and/or metal coordinated linker.

As used herein, the term “liposome” includes any structure composed of alipid bilayer that encloses one or more volumes, wherein the volume canbe an aqueous compartment. Liposome consists of one or more lipidbilayers including but not limited to phosholipid bilayer or bilayer ofnonionic surfactant. Liposomes consisting of a phospholipid bilayer maycomprise naturally-derived phospholipids with mixed lipid chains (likee.g. phosphatidylethanolamine) but are not limited to these components.Liposomes include, but are not limited to, emulsions, foams, micelles,exosomes, vesicles, insoluble monolayers, liquid crystals, phospholipidsdispersions, lamellar layers and the like. The term “liposome” alsoincludes so called “stealth liposomes” which consist of water-solublepolymers (e.g. polyethylenglycol, PEG) attached to the surface ofconventional liposomes composed of a lipid mono- or bilayer that enclosea volume (e.g. so called PEGylated liposomes).

Following liposome preparation, the liposome may be sized to achieve adesired size range and relatively narrow distribution of liposome sizes.For example delivery to the brain, the liposomes should preferably beless than about 1.0 μm in diameter, more preferably 75-400 nm, morepreferably 100-200 nm, which allows the liposome suspension to besterilized by filtration. Methods of coupling peptides to liposomesaccording to the present invention may involve either covalentcross-linking between a liposomal lipid and a peptide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic illustration of the polypeptide conjugate.

FIG. 2 is schematic illustration of a self-aggregation model of FAM-CGYnanoparticles using ChemBioOffice software where the polypeptide islinked to FAM. It is further illustrated how the peptide conjugateself-assemble into a nanoparticle or a fiber form through the formationof disulphide bridges, hydrogen bonding, possible salt-bridges andhydrophobic interaction including π-π interactions.

FIG. 3A represents nanoparticle size analysis by Nanosight ParticleTracking (NTA) of different concentrations of FAM-CGY peptide in MQwater. Corresponding representative NTA video frame is also shown (B).This is in accord with the critical aggregation concentration ofFAM-CGY, which is 2.5 micromolar and shown in FIG. 6A. FIG. 3B showsrepresentative NTA video frames of nanoparticle scattering from particlesize analysis in FIG. 3A. The arabic numeral on the down-left corner ofthe video frame represent the count number of scattered particles withinthe analysed frame.

FIG. 4 is a graph showing the relation between the peptideconcentration, particle concentration and size. The CGY-peptide is withand without FAM at the N-terminal.

FIG. 5 is a graph showing the effect of pH on size and concentration ofthe FAM-CGY-peptide-aggregates. A 5 μM FAM-CGY peptide working solutionswas prepared in 10 mM HEPES buffer, pH values were adjusted with 1M HCLor 1M NaOH.

FIG. 6. Characterization of FAM-CGY peptidic nanoparticles FIG. 6A.critical aggregation concentration measurement, FIG. 6B. atomic forcemicroscopy (AFM) representation of aggregated FAM-CGY peptides showing anetwork of globular and fibre structures, FIG. 6C. graph of particlesize distribution measured by NTA, and FIG. 6D. far-UV CD spectra ofFAM-CGY peptide at various pH.

Live-cell fluorescence microscopy of hCMEC/DE3 cells uptake of differentvariations of FAM-labeled CGY-peptides. FIG. 7A. 10 μM 5′-FAM (negativecontrol), FIG. 7B. 5 μM 5′ labeled CGY-peptide (FAM-CGY), FIG. 7C. 5 μM3′ labeled CGY-peptide (CGY-FAM), FIG. 7D. 5 μM FAM-d-CGY, FIG. 7E. 5 μMFAM-CGY scrambled 1, FIG. 7F. 5 μM FAM-CGY scrambled 2. The hCMEC/DE3cells were incubated with different variations of FAM-labeledCGY-peptides for 24 h, and washed 3 times with PBS. The nucleus wasstained with Hoechst 33342 (5 μg/mL), insert bars=50 μm.

FIG. 8 Single- and live-cell fluorescence microscopy showing hCMEC/DE3cells uptake of different variations of FAM-labeled CGY-peptides. Thecells were stained with CellLight® Actin-RFP and Hoechst 33342. Insertbars=20 μm.

FIG. 9 Quantification of FAM labeled CGY-peptide uptake in differentcell lines by fluorescence-activated cell sorting (FACS). The cells wereincubated with 5 μM of different variations of FAM-labeled CGY-peptidesat 37° C. for 24 h.

Dose and time-depended vesicular uptake of FAM-CGY peptide in hCMC/DE3cells. Cells were treated with various concentrations of FAM-CGY peptideranging from 0-20 μM and stained with Hoechst 33342. FIG. 10A.Fluorescence microscopy after 24 h. FIG. 10B. hCMC/D3 cells weresubjected to 5 μM FAM-CGY nanoparticles and studied with fluorescencemicroscopy at different time-intervals from 0-48 hours. and FIG. 10C.Quantification of peptide uptake from (a) by FACS, and FIG. 10D.quantification of uptake from (c) by FACS. The cells were stained withCellLight® Actin-RFP and Hoechst 33342, Insert bars=20 μm.

Influence of various endocytic inhibitors on the hCMEC/D3 cells uptakeof FAM-CGY peptidic nanoparticles. FIG. 11A. FACS analysis of hCMEC/D3cells incubated with FAM-CGY peptidic nanoparticles or transferrin inthe presence different inhibitors. The graph displays mean fluorescenceintensities of one of three independent experiments performed induplicate. The FAM-d-CGY and transferrin were also chosen as controls toperform the inhibition experiments. FIG. 11B. Shows the influence ofmorphology on the hCMEC/D3 cell uptake of FAM-CGY peptidic nanoparticlesin presence of various inhibitors by fluorescence microscopy. Energydependent inhibitor: 1 mM 2-deoxy-D-glucose (DOG) and 1 mM NaN3.Macropinocytosis inhibitor: 10 μM worthmanin (WOR). Fluid phaseendocytosis inhibitor: 20 μM nocodazole (NOC). Clathrin-dependentinhibitor: 30 μM chlorpromazine (CPZ). Caveolae-medicated inhibitor: 5μM N-ethylmaleimide (NEM). Caveolae-dependent endocytosis: 200 μMindomethacin (IMC). Cell nucleus were stained with Hoechest 33342,insert bars=25 μm.

FIGS. 12A, 12B, and 12C are graphs illustrating a complement activationexperiment. The complement activation products FIG. 12A. SC5d-9, FIG.12B. C5a, and FIG. 12C. C3a were quantified in healthy human serum afterincubation of CGY, FAM-CGY and FAM-d-CGY peptide at low and highconcentration. Background noise and positive control (Zymosan) arepresented for each product. Complement activation and fixation is afundamental process contributing to macrophage clearance ofintravenously injected nanopaticles. Accordingly, complement activationstudies in human serum were performed with low and high concentrationsof FAM-CGY peptide and other peptides (CGY, FAM-d-CGY) for comparison.The FAM-CGY nanoparticles had no significant effect on the level ofcomplement activation products SC5d-9, C5a, and C3a. The responses wererelatively low, and close to the background activation. Only a slightlevel of complement activation was observed compared to the positivecontrol (Zymosan). There were no systematic differences between theactivation below and above CMC. Conclusion: The FAM-CGY nanoparticlesare unlikely to induce complement-mediated immune reactions followingintravenous injection.

FIG. 13 is a Western blot and chart showing the quantification of theTFRC expression in different cell lines: human brain endothelial cellline (hCMEC/D3), human mammary Epithelial Cell Line (MCF-10A), humanbreast cancer cell line (MCF-7), human epithelial carcinoma cell line(HeLa), human umbilical vein endothelial cells (HUVEC) and human lungcarcinoma cells (H1229).

FIG. 14 is chart showing the uptake of FAM-CGY quantified by FACS. MCF-7and MCF-10A cells were incubated with different concentration of FAM-CGYas indicated for 24 h and studied by fluorescence microscopy (notshown).

FIG. 15 is a chart showing the level of competition for TFRC binding ofFAM-CGY nanoparticles and different concentrations of transferrin (62.5,250 nM or 500 nM) analyzed by fluorescence microscopy (not shown) andquantified by FACS.

FIGS. 16A and 16B show blockage of FAM-CGY nanoparticle uptake afterknocking down the TFRC expression. FIG. 16A shows a downregulated TFRCexpression after using a commercial transfection reagent siPORTAmine/TFRC siRNA complex for 72 h in hCMEC/D3 cells and a unspecificsiRNA (siControl) as a positive control. FIG. 16B shows blockage ofFAM-CGY nanoparticle uptake in TFRC knocked down hCMEC/D3 cells. Thecells with low TFRC expression and siControl transfection cells wereincubated with 5 μM FAM-CGY nanoparticles and 62.5 nM transferrin(positive control) for 16 h, respectively, the FAM-CGY nanoparticles andtransferrin uptake were detected by fluorescence microscopy (not shown)and b. quantified by FACS. Cell nucleus stained with Hoechest 33342,Insert bars=50 μm.

FIGS. 17A through 17G show FAM-CGY nanoparticles competed with differentconcentrations of RAGE receptor substrate or amyloid-β peptide. FIGS.17A through 17G were analyzed by fluorescence microscopy. FIG. 17H isquantified by FACS.

FIG. 18A shows downregulated RAGE expression using a commercialtransfection reagent siPORT Amine/RAGE siRNA complex for 72 h inhCMEC/D3 cells and the unspecific siRNA (siControl) as control. FIG. 18Bshows blockage of the FAM-CGY nanoparticle uptake in RAGE knocked downhCMEC/D3 cells. The RAGE low expression cells and siControl transfectioncells were incubated with 5 μM FAM-CGY nanoparticles and 250 nMamyloid-β peptide (positive control) for 16 h respectively, the FAM-CGYnanoparticles and amyloid-β peptide uptake was detected by fluorescencemicroscopy (not shown).

FIGS. 19A through 19C show FAM-CGY nanoparticles co-competed withtransferrin, RAGE peptide or amyloid-β peptide. FIG. 19A was analyzed byfluorescence microscopy and FIGS. 19B and 19C were quantified by FACS.

FIG. 20 Real-time trafficking FAM-CGY nanoparticles endocytosis.hCMEC/D3 cells were stained with wheat germ agglutinin (WGA)-TexasRed®-X and Hoechest 33342. Insert white frames are image amplifications.The arrows indicate the peptidic nanoparticles binding to the membraneof the cell surface or having just crossed the cell membrane. Insertbars=10 μm.

FIG. 21 Schematic illustration of the internalization mechanism of theFAM-CGY nanoparticles in hCMEC/D3 cell based on fluorescence microscopyexperiments using CellLight® Early endosomes-RFP, Lysosomes-RFP,Golgi-RFP, and Endoplasmic reticulum (ER)-RFP to stain the cellorganelles (not shown). Peptidic nanoparticle binds 1 to the cellsurface with high affinity. After being internalized by transferrinreceptor-mediated endocytosis 2 in early endosomes, the peptidicnanoparticles were transported from the early endosomes to the lysosomesand then released 4 into the cytosol. Experiments were unclear ifpeptidic nanoparticles were transported within the ER and Golgiapparatus.

FIGS. 22A and 22B are characterizations of siRNA/FAM-CGY complexes.

FIG. 22A NTA of siRNA/FAM-CGY complexes. FIG. 22B. Size distribution ofsiRNA/FAM-CGY complexes measured of NTA.

FIG. 23 Transmission electron microscopy image of FAM-CGY/Cy3-siRNAfiber complexes with an average length of 297±87 nm and width of 50±11nm.

FIG. 24 Transmission electron microscopy image FAM-CGY/ciRNA (circularRNA) nanoparticles complexes with an average diameter of 180±29.

Transfection efficiency studies of siRNA/FAM-CGY complexes. FIG. 25A.Suppression of transferrin receptor expression by different amounts ofTFRC siRNA with or without complex formation with FAM-CGY. Transferrinreceptor expression was measured 72 h after transfection. FIG. 25B. Timedependent downregulation of the transferrin receptor by TFRCsiRNA/FAM-CGY complexes.

Cell viability assessment following treatment of cells with thefluorescent peptide FIG. 26A or siRNA/FAM-CGY complex FIG. 26B for 24 h,determined by LDH assay (n=6). Background: untreated cells. To examinethe cytotoxicity of the peptide and the siRNA/FAM-CGY complex, thehCMEC/D3 cells were incubated with concentrations 1, 2, 5, 10, 20 μM ofthe peptide and different relations of siRNA/FAM-CGY complexes (8:5,16:5 or 24:5) for 24 h, the viability of cells without treatment wasused as a control. The cytotoxicity was measured by a LDH assay. The LDHassay measures the membrane integrity as a function of the amount ofcytoplasmic LDH leaked into the medium.

Investigation of oxygen consumption rate (OCR) in hCMEC/D3 cellsfollowing incubation with FAM-CGY peptide or siRNA/FAM-CGY complex. ThehCMEC/D3 cells were incubated with concentrations 2, 5, 10 or 20 μM ofthe FAM-CGY peptide for FIG. 27A 8 hours, or FIG. 27B 24 hours, or FIG.27C different formulation of siRNA/FAM-CGY complex for 24 hours. OCR wasmonitored in real-time using XF Analyzer (Seahorse Bioscience) and datawas thereafter corrected for cell numbers. Untreated cells and cellsincubated with different dilutions (100-fold or 300-fold) of Amine orsiRNA/Amine group were used as controls. FIG. 27D Standard curve showingthe linear relationship between absorbance values from crystal violetstaining and hCMEC/D3 cell numbers.

Blockage of amyloid-β peptide uptake by FAM-CGY nanoparticles analyzedby fluorescence microscopy (not shown) and quantified by FACS. FIG. 28Ashows a diagram of the number of green cell counts of cells havingFAM-CGY bound.

FIG. 28B shows a diagram of the number of red cell counts of cellshaving amyloid-β bound.

FIG. 29A represents schematic illustration of an in vitro set up forphoto-activation of FAM-CGY. The light with designated wavelength isshone from the above using a halogen light source. The dish containcells with internalised FAM-CGY nanoparticles. These particles areinternalised within endo-lysosomal compartments. On photo activation,FAM-CGY destabilises endosomes and FAM-CGY conjugates are released intothe cytoplasm. FIG. 29B represents a schematic illustration of an invitro method light-triggered release of FAM-CGY nanoparticles fromcompartment vesicles to the cytoplasm using a specific wavelength oflight. b. Fluorescence microscopy image of hCMEC/D3 cells exposed to 488nm light at time points 0, 2, 2.30, 4 or 6 minutes. The cells wereincubated with FAM-CGY nanoparticles for 24 h at 37° C.

FIG. 30 Light-triggered release of FAM-CGY nanoparticles fromcompartment vesicles to cytoplasm. hCMEC/D3 cells exposed to the 488 nmlight at 0, 4, 8, 10, 11, 13 or 14 minutes. The cells were incubatedwith FAM-CGY nanoparticles for 24 h at 37° C. prior light exposure.

FIG. 31 Fluorescence microscopy image of energy dependent controlledrelease of FAM-CGY nanoparticles from compartment vesicles to cytoplasm.hCMEC/D3 cells were exposed to differential interference contrastmicroscopy (DIC), and wavelengths of 488 (green), 405 (blue) or 635(red) nm at different times; 0, 4, 5 or 6 min for the green light, 0 or30 for DIC, 0, 4, 12 or 20 min for blue light and 0 or 30 for red light.Due to lack of light-triggered release after DIC and red light, thosecells were following exposed to green light for 4 and 6 minutes torelease the FAM-CGY nanoparticles. All cells were incubated with FAM-CGYnanoparticles for 24 h at 37° C. prior light exposure.

FIGS. 32A, 32B, and 32C are fluorescence microscopy images of in vitrolight-triggered release of siRNA by FAM-CGY nanoparticles into thecytoplasm. The fluorescence intensity of the FAM-CGY peptide, Cy3-siRNAand Hoechest 33342 (nucleus stain) in the cytoplasm is measured alongthe white line in the image. The hCMEC/D3 cells were incubated withCy3-siRNA/FAM-CGY photosensitive nanoparticle complex at 37° C. for 24 hand exposed to the 488 nm light at 0, 4, and 6 minutes.

FIG. 33A Sequence of FAM-CGY and FAM-GYR-GYR peptide sequence of a.FAM-CGY peptide and FIG. 33B FAM-GYR-GYR, and corresponding molecularenergy-minimization models using Chem BioOffice software.

FIGS. 34A and 34B are fluorescence microscopy images of thedose-depended vesicular uptake of FAM-CGY and FAM-GYR-GYR peptide inhCMEC/D3 cells. The cells were treated with concentrations of 1, 2, 5 or10 μM of FAM-CGY and FAM-GYR-GYR and stained with Hoechest 33342 priorfluorescence microscopy after 24 h (a) (Insert bars=25 μm).

FIG. 35 Comparison of the uptake of FAM-CGY and FAM-GYR-GYR peptide byhCMEC/D3 cells. Cells were treated with concentrations of 1, 2, 5 or 10μM of FAM-CGY and FAM-GYR-GYR and the peptide uptake were quantified byFACS.

FIG. 36 Influence of various endocytosis inhibitors on the hCMEC/D3cells uptake of FAM-GYR-GYR peptide. FACS analysis of hCMEC/D3 cellsincubated with FAM-GYR-GYR peptide in the presence inhibitors: Energydependent inhibitor: 1 mM 2-deoxy-D-glucose (DOG) and 1 mM NaN3.Macropinocytosis inhibitor: 10 μM worthmanin (WOR). Fluid phaseendocytosis inhibitor: 20 μM nocodazole (NOC). Clathrin-dependentinhibitor: 30 μM chlorpromazine (CPZ). Caveolae-medicated inhibitor: 5μM N-ethylmaleimide (NEM). Caveolae-dependent endocytosis: 200 μMindomethacin (IMC). The graph displays mean fluorescence intensities ofone of three independent experiments performed in duplicate.

FIGS. 37A, 37B, 37C and 37D are fluorescence microscopy images showingintracellular trafficking of FMA-GYR-GYR peptide in different organellesafter 4 h incubation. The hCMEC/D3 cell organelles were labelled withCellLight® Early endosomes-RFP, Lysosomes-RFP, Golgi-RFP, andEndoplasmic reticulum (ER)-RFP, respectively, and the nuclei was stainedwith Hoechest 33342.

FIG. 38 Fluorescence microscopy image of Cy3-siRNA/FAM-GYR-GYR complextransfection of hCMEC/D3 cells. The cells were incubated withCy3-siRNA/FAM-GYR-GYR complex as indicated at 37° C. for 1 h or 4 h. Thecell nucleus were stained with Hoechest 33342.

Bar chart showing binding of F-liposome alone, F-Liposome-CGY,F-Liposome-FAM-CGY, F-Liposome-CGY-FAM or F-Liposome-FAM-CGY-scrabled2conjugates to hCMEC/D3 cells. The cells were analysed by FACS. Theliposomes in FIG. 39A were labeled with RED Fluorescent phospholipid andin FIG. 39B with green Fluorescent phospholipid.

FIG. 40 is a graph showing size distribution of caprylic acid conjugatedCGY peptide as measured by nanoparticle tracking analysis (NTA).

FIG. 41-44 are results of studies illustrating formation of peptidiccomplexes based on rhodamine-linked CGY (Rh-CGY) peptides.

FIG. 41A shows a typical size distribution profile of Rh-CGY peptide (10μM) self-assembly determined by NTA FIG. 41B shows the uptake of Rh-CGYpeptide self-assembly in hCMEC/D3 cells.

FIG. 42 is a Western blot showing peptidic complexes in a study ofRh-CGY.

FIG. 43A is a histogram representing a typical size distribution profileof complex species of Rh-CGY (10 μM) with FAM-(C)-NAP peptide (10 μM)determined by NTA. FIG. 43B shows the morphology of Rh-CGY/FAM-(C)-NAPpeptidic complex observed by electron microscopy. The Rh-CGY/FAM-(C)-NAPpeptide co-self assembles into nanoparticles and fibers. FIG. 43C showsthe intracellular intensity of cargoes delivered into hCMEC/D3 cells bythe Rh-CGY peptidic based nanosystems using FASC analysis. FIG. 43D is atypical dot plot resulting from a fluorescence-assisted cell sortingexperiment of Rh-CGY and FAM-(C)-NAP peptide after treating hCMEC/D3cells with 10 μM FAM-(C)-NAP, 10 μM Rh-CGY/10 FAM-(C)-NAP complex and 10μM Rh-CGY for 24 h. FIG. 43E is a photograph of living cell microscopyimages of the cells from the study of FIG. 43D.

FIG. 44A represents typical size distribution profile of Rh-CGY (10μM)/FAM-GYR (5 μM) peptidic complex determined by NTA. FIG. 44B showsthe morphology of Rh-CGY/FAM-GYR peptidic complex observed by electronmicroscopy.

FIG. 44C is a typical dot plot resulting from a fluorescence-assistedcell sorting experiment of Rh-CGY and FAM-GYR peptide after treatinghCMEC/D3 cells with 5 μM FAM-GYR, 10 μM Rh-CGY/5 μM FAM-GYR complex and10 μM Rh-CGY for 24 h. FIG. 44D is a photograph of live cell microscopyimages of the cells from the study of FIG. 44C.

FIG. 45 shows photographs of hCMEC/d3 cells in a study investigatingdouble peptide-mediated nucleic acid transfection.

FIG. 46 shows fluorescence imagery of organs from mice involved in invivo studies of peptide localization.

FIG. 47 are photographs of brain cells of mice that were involved in invivo studies of peptide conjugate localization.

FIGS. 48A through 48G demonstrate the structures of peptide conjugateaggregates. FIGS. 48A and 48B are atomic force microscopy (AFM) imagesof FAM-CGY (5 μM) species in the absence and presence of 24 nM siRNA (inthis case, targeted against the transferrin receptor), respectively. Theinset in FIG. 48B is transmission electron micrograph ofelliptically-shaped FAM-CGY/siRNA complexes showing the presence of someelongated fibre-like structures in the particle core. The sphericalequivalent mean size of nanoparticles is 169±51 nm, based onmeasurements of at least 100 individual images. FIGS. 48C, 48D, 48E,48F, and 48G show an overview of peptide nanoparticle-fiber network(48C), a typical core-shell nanoparticle component of the network (48D),a core-shell nanoparticle with elongated hair-like projections (48E),and magnified views of twisted fibre components (48F, 48G). The measuredmean size of the core-shell nanoparticles were 116±22 nm (based onmeasurements of at least 100 randomly selected nanoparticle images.)

DETAILED DESCRIPTION

This section describes the current invention in greater detail using aschematic illustration of a polypeptide conjugate and examples ofpolypeptide conjugates and their use in a method to for binding and/orinternalization of the polypeptide conjugate to a mammalian cell havinga transferrin receptor (TFRC) and/or receptor for advanced glycation endproducts (RAGE). However, this by no means limits the scope of thecurrent invention.

FIG. 1 shows a schematic drawing of a polypeptide conjugate with amoiety 1 and connected to a linker 2. A second moiety 5 may be linked tothe linker 2. The linker is also linked to polypeptide sequenceGly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly 3 which maybe connected to one or more polypeptides 3 by a spacer 4.

EXAMPLES Example 1: Self-Assembly of Polypeptide Conjugate

Scrambled peptides and fluorescence-labelled peptides were designed andsynthesized as shown in table 1. To investigate whether the polypeptideconjugate can self-assemble, Nanoparticle Tracking Analysis (NTA)technology was employed to detect the peptide aggregation. Briefly, 1 mLpeptide solutions with different concentrations (0.5, 1, 2.5 and 5 μM)were prepared by diluting the peptide stock solutions (500 μM) with MQwater and incubate for 30 min at room temperature. NTA measurements wereperformed with a NanoSight LM20 (NanoSight Ltd., Amesbury, UnitedKingdom) equipped with a sample chamber with a 405 nm blue laser and aViton fluoroelastomer O-ring. The peptide samples were injected in thesample chamber with sterile syringes (BD Discardit II, New Jersey, USA)until the liquid reached the tip of the nozzle. All measurements wereperformed at room temperature. For the pH effect of FAM-CGY peptideassembly, FAM-CGY stock solution (500 μM) was diluted into 5 μM with 10mM HEPES buffer where pH values were adjusted with 1 M HCl or 1 M NaOH,respectively.

FAM-CGY self-assembles into nanoparticles and fibres as shown in FIG. 2.The size of the self-assembled FAM-CGY nanoparticles are 2-200 nmdepending on the peptide conjugate concentration.

TABLE 1 SEQ ID Molecular Peptide Modified site Peptide sequence NO.weight Purity CGY Unlabel peptide Cys-Gly-Tyr-Arg-Pro- 3 1820.04 98.11%Val-His-Asn-Ile-Arg-Gly- His-Trp-Ala-Pro-Gly FAM-CGY Original peptide5-FAM-Cys-Gly-Tyr-Arg- 4 2178.36 98.91% Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly CGY-FAM 5-FAM C-terminal Cys-Gly-Tyr-Arg-Pro- 52305.53 98.36% peptide, 5-FAM Val-His-Asn-Ile-Arg-Gly- effectHis-Trp-Ala-Pro-Gly-Lys- 5-FAM FAM-d-CGY D form amino acid5-FAM-dCys-dGly-dTyr- 6 2177.36 98.34% affect dArg-dPro-dVal-dHis-dAsn-dIle-dArg-dGly- dHis-dTrp-dAla-dPro- dGly FAM-CGY Change the5-FAM-Cys-Gly-Tyr-Arg- 7 2177.36 98.56% Scrambled 1 position of Arg, thePro-Val-His-Asn-Ile-Gly- charge effect His-Trp-Arg-Ala-Pro-Gly FAM-CGYWithout 5-FAM-Cys-Gly-Tyr-Arg- 8 2048.20 98.26% Scrambled 2 Tryptophan,Pro-Val-His-Asn-Ile-Arg- relative the Gly-His-Gly-Ala-Pro-GlyTryptophan effect

Example 2: Specific Binding to and Cellular Uptake of FAM-LabeledCGY-Peptide by hCMEC/D3 Cells

To evaluate the role of the transferrin receptor in the uptake ofFAM-CGY nanoparticles, hCMEC/D3 cells were incubated with bothtransferrin and FAM-CGY nanoparticles in competition experiment (FIG.15).

The determination of fluorescence peptide internalization in hCMEC/D3cells by flow cytometry (FACS) was performed as follows. Cells(2×10⁴/cm²) were seeded on 24-well plate (Corning, N.Y.) and grown 2days at 37° C. and 5% CO₂ in order to reach 60%-70% confluency. Thecells were washed 3 times with pre-heated PBS and 200 μL of 5 μMfluorescence-labelled peptides (diluted in cell medium containing serum)was added. After 24 h of incubation at 37° C. with 5% CO₂, each chamberwas washed with pre-heated PBS 3 times and incubated with fresh cellgrowth medium. The cell nucleus was stained with Hoechst 34580 dye (5ug/mL), cell membrane was stained with Texas Red®-X wheat germagglutinin (5 ug/mL). FAM-CGY nanoparticles competed with differentconcentrations of transferrin or added with the peptides in differentconcentrations ranging from 62.5-500 nM and were analyzed byfluorescence microscopy and quantified by FACS.

The live cell imaging was performed on a widefield microscope (LeicaAF6000LX, Germary) using a 63× oil objective with 1.6 magnification andfilters GFP (Ex BP 470/40, Em BP 525/50), Cy3 (Ex BP 555/25, Em BP605/52) and A4 (Ex BP 360/40, Em 470/40). Treated cells were then washed3 times with pre-warmed PBS, and harvested by trypsinization. A total of10,000 cells were analyzed by flow cytometry (FACS Array™ Cell Analysis,BD, USA).

The competition experiment shows that the uptake of the FAM-CGYnanoparticles is significantly decreased with increasing transferrinconcentration suggesting that FAM-CGY nanoparticles strongly competewith transferrin on binding to the transferrin receptor.

Example 3: Specific Binding and Cellular Uptake of FAM-LabeledCGY-Peptide to hCMEC/D3 Cells

To evaluate the role of the RAGE in the uptake of FAM-CGY nanoparticles,hCMEC/D3 cells were co-treated with RAGE-peptide or amyloid-8 andFAM-CGY nanoparticles in competition experiments (FIG. 17).

The determination of fluorescence peptide internalization in hCMEC/D3cells by flow cytometry (FACS) was performed as follows. Cells(2×10⁴/cm²) were seeded on 24-well plate (Corning, N.Y.) and grown 2days at 37° C. and 5% CO₂ in order to reach 60%-70% confluency. Thecells were washed 3 times with pre-heated PBS and 200 μL of 5 μMfluorescence-labelled peptides (diluted in cell medium containing serum)was added. After 24 h of incubation at 37° C. with 5% CO₂, each chamberwas washed with pre-heated PBS 3 times and incubated with fresh cellgrowth medium. The cell nucleus was stained with Hoechst 34580 dye (5ug/mL), cell membrane was stained with Texas Red®-X wheat germagglutinin (5 ug/mL). FAM-CGY nanoparticles (5 μM) competed withdifferent amounts of RAGE-peptide or amyloid-8 peptide (5, 10 or 20 μg)and were analyzed by fluorescence microscopy (data not shown) andquantified by FACS (FIG. 17).

The live cell imaging was performed on a widefield microscope (LeicaAF6000LX, Germary) using a 63× oil objective with 1.6 magnification andfilters GFP (Ex BP 470/40, Em BP 525/50), Cy3 (Ex BP 555/25, Em BP605/52) and A4 (Ex BP 360/40, Em 470/40). Treated cells were then washed3 times with pre-warmed PBS, and harvested by trypsinization. A total of10,000 cells were analyzed by flow cytometry (FACS Array™ Cell Analysis,BD, USA).

The competition experiment shows that the uptake of the FAM-CGYnanoparticles is significantly decreased with increasing RAGE-peptide oramyloid-β concentration suggesting that FAM-CGY nanoparticles stronglycompete with the RAGE-peptide and amyloid-β on binding to the RAGEreceptor.

Example 4: TFRC Gene Knock-Out Experiments

The transferrin receptor (TFRC) expression was downregulated by using acommercial transfection reagent siPORT Amine/TFRC siRNA complex to studythe possible blockage of the FAM-CGY nanoparticle uptake in hCMEC/D3cells. The commercial transfection reagent siPORT Amine/TFRC siRNAcomplex was incubated for 72 h in hCMEC/D3 cells including an unspecificsiRNA (siControl) as control. The TFRC low expression cells andsiControl transfection cells were incubated with 5 μM FAM-CGYnanoparticles and 62.5 nM transferrin (positive control) for 16 h,respectively. The FAM-CGY nanoparticles and transferrin uptake wasdetected by fluorescence microscopy (not shown) and FACS (FIG. 16).

FAM-CGY nanoparticle uptake is markedly reduced in TFRC knocked outhCMC/DE3 cells to nearly the same level the as transferrin uptake.Hence, TFRC functions as a receptor for FAM-CGY nanoparticle binding anduptake.

Example 5: RAGE Gene Knock-Out Experiments

The RAGE expression was downregulated by using a commercial transfectionreagent siPORT Amine/RAGE siRNA complex to study the possible blockageof the FAM-CGY nanoparticle uptake in hCMEC/D3 cells. The commercialtransfection reagent siPORT Amine/RAGE siRNA complex was incubated for72 h in hCMEC/D3 cells including an unspecific siRNA (siControl) ascontrol. The TFRC low expression cells and siControl transfection cellswere incubated with 5 μM FAM-CGY nanoparticles and 250 nM amyloid-8(positive control) for 16 h, respectively. The FAM-CGY nanoparticles andRAGE peptide uptake was detected by fluorescence microscopy (not shown)and FACS (FIG. 18).

FAM-CGY nanoparticle uptake is markedly reduced in RAGE knocked outhCMC/DE3 cells to nearly the same level as amyloid-β uptake. Hence, RAGEfunctions as a receptor for FAM-CGY nanoparticles.

Example 6: Formation of siRNA/FAM-CGY Complex

Different molar rates of siRNA were added to the FAM-CGY peptidesolution (50 μM/L) dropwise in saline solution and incubated for 30 min.Then the mixture solution was diluted with serum free hCMEC/D3 cellmedium to a final peptide concentration 5 μM. The atomic forcemicroscopy (AFM) (FIG. 22a ), nanoparticle tracking analysis (NTA) (FIG.22b ), fluorescence microscopy (not shown), dynamic light scattering(DLS) technology (not shown) and transmission electron microscopy (TEM)(FIG. 23) were employed to characterize the siRNA/FAM-CGY complex.Complexes with circular RNA (ciRNA) and FAM-CGY was also studied usingTEM (FIG. 24). Briefly, one drop of the final Cy3-siRNA/FAM-CGY solutionwas added on microscope glass slides (VWR) and left to air-dry at roomtemperature for 30 min. The Cy3-siRNA/FAM-CGY complexes were imaged byfluorescent microscopy (Leica AF6000LX, Germany) using a 100×TIRF oilobjective with 1.6 magnification and filters GFP (Ex BP 470/40, Em BP525/50), and Cy3 (Ex BP 555/25, Em BP 605/52). The size and surfacecharge of FAM-CGY peptidic nanoparticles and siRNA/FAM-CGY complex wereinvestigated using a Zetasizer Nano ZS goniometer (Malvern Instruments,Malvern, UK) containing a He—Ne laser source (A=633 nm, 22 mW outputpower). All the measurements were carried out at 25° C. Threemeasurements each with 30 sub-runs were performed for each sample andresults are shown in Table 2.

FAM-CGY forms stable spherical and rod shaped nanoparticles with siRNAhaving average diameter of 168±12 nm for spherical particles analyzed byNTA and for fibres a length of 297±87 nm analysed by TEM 23), which isslightly larger than FAM-CGY nanoparticles described above. Theseresults are similar to dynamic light scattering (DLS) characterization,where the siRNA/FAM-CGY particles are in the size range of 170-180 nmand display a slightly positive surface charge with zeta potentialsranging from 5.57±0.87 to 1.27±0.38 mV.

TABLE 2 The size and zeta potential of FAM-CGY nanopaticles andsiRNA/FAM-CGY complexes characterized by dynamic light scattering (DLS).Zeta potential Samples Size (nm) PDI (mV) 5 μM FAM-CGY 156.6 ± 2.1 0.325± 0.028 7.27 ± 0.73 nanopartilces 8 nM siRNA/5 μM 170.2 ± 5.3 0.380 ±0.033 5.57 ± 0.87 FAM-CGY complex 16 nM siRNA/5 μM 173.8 ± 3.2 0.402 ±0.047 2.87 ± 0.50 FAM-CGY complex 24 nM siRNA/5 μM  180.6 ± 12.1 0.397 ±0.054 1.27 ± 0.38 FAM-CGY complex

Example 7: Transfection of hCMC/DE3 Cells with Cy3-siRNA/FAM-CGY Complex

To confirm the rate of cellular transfection, different molar ratios ofCy3-fluorescence labeled siRNA (Cy3-siRNA) were incubated with FAM-CGYpeptide to form polyplexes, and added to hCMEC/D3 cells (data notshown). Different molar rates of Cy3-siRNA were added to FAM-CGY peptidedropwise in saline solution and incubated for 30 min. Then the mixturesolution was diluted with normal hCMEC/D3 cells medium to a finalconcentration of FAM-CGY peptide 5 μM (5000 nM). The cells wereincubated with different rates of Cy3-siRNA/FAM-CGY complex as indicatedat 37° C. for 24 h. The cell nucleus was labelled with Hoechest 33342(blue), insert bars=50 μm.

The Cy3-siRNA/FAM-CGY complex exhibits significant cellular uptake byhCMEC/D3 cells in all molar ratios compared to Cy3-siRNA alone.

Example 8: Transfection of hCMC/DE3 Cells with Cy3-siRNA/FAM-CGY Complex

The siRNA efficacy was investigated by subjecting hCMEC/D3 cells tofunctional TFRC siRNA/FAM-CGY complexes (FIG. 23).

The intracellular distributions of siRNA complexed with the FAM-CGYnanoparticles were observed by fluorescent microscopy usingCy3-fluorescence labeled siRNA. In vitro gene silencing experiments wereperformed in hCMEC/D3 cells using the TFRC siRNA and a scrambled siRNAas a negative controls. The level of TFRC expression in hCMEC/D3 cellstreated with different siRNA/FAM-CGY formulations were investigated bywestern blot.

Cells treated with the scrambled siRNA complexes do not give rise to anysignificant gene silencing even when using up to 24 nM siRNA mixed withFAM-CGY or siPORT Amine (Amine). In contrast, TFRC gene expression isslightly reduced by TFRC siRNA/FAM-CGY complexes containing 8 nM siRNA.When using TFRC siRNA up to 24 nM, the TFRC siRNA/FAM-CGY complexesefficiently inhibit TFRC gene expression down to 32.4% after 72 h oftransfection. The siRNA/FAM-CGY delivery system showed a bettersilencing efficiency than siRNA/Amine which leads to 45.5% TFRCknockdown. Moreover, the siRNA/FAM-CGY complex transfection is timedependent. After 24 hours and 48 hours, only 20% gene expressionknockdown is detected, while after 72 hours significantly more knockdownis observed.

Example 9: Cytotoxicity of Cy3-siRNA/FAM-CGY

The cytoxicity of FAM-CGY peptide and siRNA/FAM-CGY complex wasevaluated by lactase dehydrogenase (LDH) assay (FIG. 26). The hCMEC/D3cells were incubated with different concentrations of the peptide (from1 to 20 μM) and different formulations of siRNA/FAM-CGY complexes for 24h. The viability of cells without treatment was used as a control. TheLDH assay has means of measuring the membrane integrity as a function ofthe amount of cytoplasmic LDH leaked into the medium. Compared to thecontrol in FIG. 26a , the LDH leakage in the medium for the groupstreated with different concentrations of the peptide did notsignificantly increase. Thus, there were no significant differencesobserved between the groups treated with FAM-CGY, FAM-d-CGY and CGY-FAM.A 30% LDH leakage was observed in cells treated with siRNA/Amine group,indicated that it was more toxic than the siRNA/FAM-CGY complexes.

These results indicate that there is no significant cytotoxicityobserved for the FAM-CGY peptide or the siRNA/FAM-CGY complexes.

Example 10: Light-Triggered Release siRNA

To evaluate the intracellular release of siRNA loaded on FAM-CGYnanoparticles by illumination, Cy3-siRNA was complexed with FAM-CGY atmolar ratio of 1:200 (Cy3-siRNA: FAM-CGY) in 50 μL saline at aconcentration of 20 μM FAM-CGY. After 30 min incubation, theCy3-siRNA/FAM-CGY complex was diluted with 150 μL 5% FBS cell medium toa final concentration of 5 μM FAM-CGY. hCMEC/D3 cells were seeded in8-well Lab-Tek chamber slides (Nunc, Naperville, Ill.) (2×10⁴/cm²) andgrown for 24 h. The cells were then washed with pre-warm PBS and treatedwith 200 μL complexes. After 24 h of incubation, the nuclei was stainedwith Hoechest 33342 (5 μg/mL) for 10 min and cells were rinsed threetimes with pre-warmed PBS. The cells were irradiated with the 488 nmlight at different time points 0, 2, 2.30, 4 or 6 minutes passed throughthe 63× objective lens, and the images were each recorded at 30 s byfluorescent microscopy using filters GFP (Ex BP 470/40, Em BP 525/50),Cy3 (Ex BP 555/25, Em BP 605/52) and A4 (Ex BP 360/40, Em 470/40),respectively. The fluorescence intensity of the FAM-CGY peptide,Cy3-siRNA and Hoechest 33342 (nucleus) in the cytoplasm along specificarea were quantified by software LAS AF Lite 6.0 (Leica, Germany) (FIG.29).

FAM-CGY is released from internalized vesicles after between 2.5 and 4minutes of light exposure.

Example 11: Preparation of Liposomes

F-Liposomes consisting of phospholipids, cholesterol, and functionalizedcoupling lipid (MPB-PE) at a molar ratio ofDPPC:Cholesterol:DSPE-PEG:DSPE-PEG-MPB at a ratio of 7:2.5:0.025:0.025were produced from lipid films hydrated with PBS. The finalconcentration was 10 μmol lipid/ml buffer. The hydration was performedin a water bath at 56° C. for 30 min. The resulting multilamellarvesicles were extruded (LiposoFast Extruder) 21 times through apolycarbonate filter (Avanti) with a pore size of 100 nm. The liposomesize was determined by NanoSight LM20 (NanoSight, Amesbury, UnitedKingdom). Liposomes were labeled with red fluorescent phospholipid (16:0Liss Rhod PE[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamineB sulfonyl) (ammonium salt)]) or green fluorescent phospholipid (18:1 PECF [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein)(ammonium salt)]).

Coupling Peptides to Liposomes

Peptides (CGY-peptide or GYR-GYR-peptide (that is a dimer ofCGY-peptide)) were reduced using 2 mM Bond-Breaker TCEP (Pierce) underNitrogen atmosphere for 1 h at 37° C. After gel filtration using asepharose CL-4B column, for removing TCEP. Reduced peptide was incubatedwith preformed maleimide-containing liposomes (The molar ratio ofphospholipids to peptide was 1 μmol to 1 nmol) under nitrogen atmosphereovernight at room temperature. Unreacted maleimide groups wereinactivated by incubation with 0.5 mM cysteine for 30 min at roomtemperature. For removing non-conjugated peptides the mixture wascentrifuged 3 times at 75.000 rpm for 30 min at 4° C. and theresuspended in PBS. Then the phospholipid concentration was againmeasured, and for indirectly measuring the amount of peptides that hasbeen bond to the liposomes, the peptide concentration in supernatant wasmeasured by Bradford assay method. Conjugated liposome was characterizedby SDS-PAGE and measuring the size by NanoSight instrument.

Uptake study of Conjugated Peptides by FACS

The cells used were human adult brain endothelial cell line hCMEC/D3were grown in endothelial growth medium 2 (EGM-2, Lonza, UK)supplemented with fetal bovine serum (FBS) 5%, hydrocortisone 1.4 μM,basic fibroblast growth factor 1 ng/mL, penstrep 1% and HEPES 10 nM in24 well tissue culture plates. The cells were used at 70% confluence(corresponding cells to 6×10⁴ to 8×10⁴ cells) were incubated withliposomes labeled with 0.2 μmol RED or GREEN fluorescently-taggedphospholipid (F-liposomes) and bearing the peptide conjugate orliposomes attached to fluorescent CGY or scrambled CGY (e.g., FAM-CGY,CGY-FAM and FAM-CGY Scrambled 2). Liposomes were added to the cells (in200 μL medium) and after overnight incubation the cells were washed with1% BSA in PBS then detached from cell culture dishes using trypsin(Sigma-Aldrich, Inc). The cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson) (FIG. 39).

F-Liposome and FAM linked to CGY-peptide or GYR-GYR-peptides in anyposition bind to hCMEC/D3 cells in a greater number than the control andF-Liposome alone.

Example 12: Double Peptide FAM-GYR-GYR Experiments

The different cellular uptake of FAM-CGY and FAM-GYR-GYR peptides inhCMEC/D3 cells was investigated by flow cytometry (FACS). hCMEC/D3 cells(2×10⁴/cm²) were seeded on 24-well plate (Corning, N.Y.) and grown 2days at 37° C. and 5% CO₂ in order to reach 60%-70% confluency. Thecells were washed 3 times with pre-heated PBS. The peptide uptakeexperiments were initiated by adding 200 μL of a range of FAM-CGY andFAM-GYR-GYR in different concentrations (1-10 μM/L, diluted in cellmedium containing serum). The double peptide contains the sequenceGly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly-Gly-Gly-Tyr-Arg-Pro-Val-His-Asn-Ile-Arg-Gly-His-Trp-Ala-Pro-Gly(SEQ ID NO.2) and optionally has an N-terminal cysteine residue.

After 24 h incubation at 37° C. with 5% CO₂, each chamber was washedwith pre-heated PBS 3 times, and harvested by trypsinization. A total of10,000 cells were analyzed by flow cytometry (FACS Array™ Cell Analysis,BD, USA) (FIG. 35).

FAM-GYR-GYR peptide/structures show considerably higher uptake byhCMEC/D3 cells compared with FAM-CGY peptide at all testedconcentrations. The double peptide shows excellent target binding, evenincreased when compared with the single sequence and allows for deliveryof larger molecules (e.g., DNA). Trials illustrated utilized a doublepeptide with a glycine residue between the two peptide sequence elementsbut other amino acid linkers, or other non-amino acid linker moleculescan be used to create such a molecule.

Example 13: Transfection of Cy3-siRNA/FAM-GYR-GYR Complex on hCMEC/D3Cells

Cy3-siRNA was complexed with FAM-GYR-GYR at a molar ratio of 1:200(Cy3-siRNA: FAM-CGY) in 50 μL saline at a concentration of 20 μM/LFAM-GYR-GYR. After 30 min incubation, the Cy3-siRNA/FAM-GYR-GYR complexwas diluted with 150 μL 5% FBS cell medium to a final concentration of 5μM/L FAM-GYR-GYR. hCMEC/D3 cells were seeded in 8-well Lab-Tek chamberslides (Nunc, Naperville, Ill.) (2×10⁴/cm²) and grown for 24 h. Thecells were then washed with pre-warm PBS and treated with 200 μLCy3-siRNA/FAM-GYR-GYR complexes. After 1 or 4 h of incubation, thenuclei was stained with Hoechest 33342 (5 μg/mL) for 10 min and cellswere rinsed three times with pre-warmed PBS. Finally, the cells wereobserved with a Leica fluorescent microscopy (Leica AF6000LX, Germary)using a 63×TIRF oil objective and filters GFP (Ex BP 470/40, Em BP525/50), Cy3 (Ex BP 555/25, Em BP 605/52) and A4 (Ex BP 360/40, Em470/40), respectively (FIG. 36).

This trial demonstrates that Cy3-siRNA/FAM-GYR-GYR complex internalizesinto hCMEC/D3 cells.

Example 14: Binding a Hydrophobic Moiety to a Peptide-FormedNanoparticle

Caprylic acid conjugated CGY peptide (Caprylic-CGY) was synthesized by asolid phase method. Peptides were purified by preparative HPLC andcharacterized by analytical HPLC and mass spectrometry (M_(w)=1946.29,Purity: 98.26%). The lyophilized peptides were dissolved into dimethylsulfoxide (DMSO) with a peptide concentration of 500 μM and stored at−80° C. For the self-assembly, the 500 μM stock solution of Caprylic-CGYwas diluted into MQ water with the final concentration of 5 μM andincubated at room temperature for 1 h. The size of self-assembly ofCaprylic-CGY was performed by Nanoparticle Tracking Analysis (NTA)(LM20, NanoSight, Amesbury, United Kingdom) with a sample chamber with a405 nm blue laser and a Viton fluoroelastomer O-ring. As seen in FIG.41, The mean size of self-assembly is 117±74 nm and with a mode of 86nm.

Example 15: Rhodamine as a Drug or Fluorescent Molecule Attached to aPeptide

Peptidic complexes were formed by drop addition of cargoes (FAM-(C)-NAP,FAM-GYR, FAM-NAP, FAM) to 80 μM rhodamine-conjugated CGY peptide(Rh-CGY) or 80 μM CGY peptide (Table X) in MilliQ (MQ) water withequatable liquid volume and incubated for 60 min. The mixture wasdiluted with MQ water to a final Rh-CGY or CGY peptide concentration of10 μM. The peptidic complexes were characterized by NTA for size andelectron microscopy (EM) for mophology. Western blot was also employedto detect disulfide bond, which form between the cargo and Rh-CGYpeptide or CGY peptide. Briefly, the peptide or complex samples wereloaded on 10% Bis-Tris mini gels (Invitrogen, Calif., USA) and subjectedto electrophoresis. The separated samples were electrophoreticallytransferred to PVDF membranes by use of an iBlot™ Gel Transfer Device(Life Technologies, USA). Membranes were blocked for 1 h at roomtemperature in 3% BSA/TBST (137 mM Sodium Chloride, 20 mM Tris, 0.1%Tween-20, pH 7.6), and incubated over night at 4° C. with HRP-Goatanti-Fluorescein antibody (diluted 1:1000 in 2% BSA/TBST). Fordetection, membranes were incubated with Novex® ECL ChemiluminescentSubstrate Reagent Kit (Invitrogen, Calif., USA).

Delivery peptide to hCMEC/D3 cells. In vitro delivery experiments wereperformed in hCMEC/D3 cells. Briefly, the peptidic complexes werefirstly prepared with concentration of 80 μM Rh-CGY or 80 μM CGYpeptide, and diluted with cell medium to 10 μM Rh-CGY or CGY peptide.The complex solutions were added to hCMEC/D3 cells following 24 hincubation. Live cell images were obtained by widefield microscopy, andthe intercellular fluorescence intensity was quantified by FACS.

To investigate the effect of hydrophobic block FAM in peptideself-assembly, another fluorophore Rhdomine B was choose to conjugatewith CGY peptide in the N-terminal, which termed Rh-CGY (molecular mass2244.69 g/mol). The measurement of NTA indicated that the Rh-CGY canalso easily self-assembly. FIG. 41 represents the size distribution ofRh-CGY supermolecular with narrow peak and a weak trailing fraction. Theaverage size of Rh-CGY supermolecular was 131±60 nm with a modal size of94 nm (FIG. 41a ). Also the supermolecular of Rh-CGY can efficientlyenter the hCMEC/D3 cells (FIG. 41b ). In order to investigate thefunctionality of Rh-CGY peptide, a model cargo of FAM-(C)-NAP peptidewas selected to co-assemble with Rh-CGY peptide. From the Western blotdata, FAM-(C)-NAP peptide form disulfide bond with Rh-CGY peptide (FIG.42). The lanes of the Western blot are as follows: Lane 1: Rh-CGYpeptide, Lane 2: FAM-(C)-NAP peptide, Lane 3: CGY peptide, Lane 4:FAM-GYR peptide, Lane 5: Rh-CGY/FAM-(C)-NAP peptidic complex, Lane 6:Rh-CGY/FAM-GYR peptidic complex, Lane 7: CGY/FAM-(C)-NAP peptidiccomplex.

Because of this disulfide bond and the π-π interaction between thefluorophore of FAM and Rhodamine, the FAM-(C)-NAP peptide can formstable complex with Rh-CGY peptide. The average size is 99±41 nm (FIG.43a ). The mixture of Rh-CGY and FAM-(C)-NAP self-assembled into notonly the nanoparticles but also into fibres based on the EM observation(FIG. 43b ). With the assistance of Rh-CGY peptide, the FAM-(C)-NAPpeptide can be easily delivered into hCMEC/D3 cells (FIG. 43 c, d, e).When FAM-(C)-NAP peptide mixed with CGY peptide, it can also form adisulfide bond with CGY peptide (FIG. 42).

However, this mixture cannot form any particles and the mixture ofFAM-(C)-NAP and CGY peptide cannot deliver FAM-(C)-NAP peptide intocells (FIG. 43c ). When cysteine amino acid was deleted from thesequence of the FAM-(C)-NAP peptide, FAM-NAP peptide was poorly taken upby hCMEC/D3 cells (FIG. 43c ). When it was co-incubated with Rh-CYGpeptide, it did not form disulfide bond with Rh-CGY peptide. FAM-NAPpeptide did not efficiently cross cell membrane even after co-incubatedwith Rh-CGY peptide (FIG. 43c ). These observations indicated thatforming disulfide bond is one of key factor to delivery FAM-(C)-NAPpeptide into the cells. This can be most readily accomplished with theinclusion of a cysteine residue, and such inclusion can be at manydifferent points of the sequence, including at the N-terminus, at theC-terminus, or internally.

In another case, FAM-GYR peptide can form stable particles/fiber complexwith Rh-CGY peptide with the mean size of 82±43 nm (FIG. 44a,b ).FAM-GYR peptide trapped in Rh-CGY peptide was significantly deliveredinto hCMEC/D3 cells compared with FAM-GYR peptide alone (FIG. 44c, d ).The FAM-GYR peptide does not contain the cysteine, and cannot formdisulfide bond to cross link with Rh-CGY peptide (FIG. 42). But the mainsequence of FAM-GYR was the same with Rh-CGY peptide, the amino acid inFAM-GYR and Rh-CGY peptide, such as Arg, Trp, can interact with eachother to form hydrogen bond and π-π interaction. This resultdemonstrated that the Rh-CGY/FAM-GYR peptidic mixture can self-assembleinto stable complexes even without the disulfide bond (FIG. 44a,b ).Cells were labelled with Hoechest 33342 (blue).

To further prove this concept, the fluorophore FAM alone wasco-incubated with the Rh-CGY peptide. FAM can not been entrapped in theRh-CGY peptidic complexes. The mixture of FAM and Rh-CGY peptide can notenhance the uptake of FAM in hCMEC/D3 cells (FIG. 43c ). This suggeststhat the sequence of FAM-GYR peptide is specific to form stableparticles with Rh-CGY peptide and improve the uptake in hCMEC/D3 cells.Rh-CGY peptide can co-assemble with the cargo peptide with specificproperties to fabricate stable nanoparticle/fiber complex because offorming disulfide bond or hydrogen bond and π-π interaction, and deliverthe cargo peptide into hCMEC/D3 cells. Rh-CGY peptide is a potentialcandidate of peptidic-based delivery system for the CNS disease.

Example 16: Plamsid Transfection Mediated by Double Peptide

To evaluate double peptide (DP)-mediated gene transfection, differentconcentrations of DP and constant amounts of pcDNA3.1NT-GFP expressionplasmid (0.3 μg) (the peptide/DNA change ration of 1:10, 1:20 and 1:40were investigated in this study) were mixed into 50 μL serum free media,and complexes were formed for 1 h at room temperature, after whichanother 150 μL serum free media was added (total volume of peptide/DNAcomplex was 200 μL). The cultured hCMEC/D3 cells (2×10⁴ cells) wereoverlaid with 200 μL peptide/DNA complex, followed by incubation for 4 hat 37° C. in 5% CO₂ atmosphere. The cultures were then a washed oncewith serum-free media and transferred to complete media containing 5%serum for growth. After 48 h, GFP gene expressions were monitored byfluorescence microscopy. Lipofectamine® 2000-mediated transfections wereperformed as described by the manufacturer's protocol (LifeTechnologies, Calif., USA).

Since the double peptide (DP) has four positive charge amine acids ineach peptide molecular, it is possible for DP to bind with DNA byelectrostatic interaction and form stable complex. In order to test thepossibility of DP-mediated gene delivery, DP was mixed with plasmid DNAencoding GFP in different charge ratios from 10:1 to 40:1. Transfectionwas performed with different peptide/DNA charge ratios in hCMEC/D3cells, and transfection efficiencies were evaluated using fluorescencemicroscopic analysis of GFP expression. The results showed that DP wasable to mediate translocation of plasmids into cells when the chargeratios of peptide/DNA more than 20 (FIG. 45). And the transfectionefficiency of peptide/DNA with charge ratio 40:1 was comparable withcommercial transfection reagent Lipofectamine® 2000 based on theintensity of fluorescence microscopy signal. But compared with the DP,the Lipofectamine® 2000, the cationic lipid based gene delivery system,can lead to higher cytotoxicity. The results indicated that the DP is apotential safe delivery system of plasmid for brain targeting. Cellswere labelled with Hoechest 33342 (blue).

Example 17: Targeting of the Brain In Vivo

BALB/c nude mice received 5 μM of FAM-CGY peptide or 10 μM of Rh-CGYpeptide (the final concentration in blood) intravenously and subjectedto optical imaging at various time points post-injection. The in vivofluorescence imaging was performed using the IVIS Imaging System 200Series and analyzed using the IVIS Living Imaging 3.0 software (CaliperLife Sciences, Alameda, Calif., USA). Optimized GFP filter or Dsredfilter sets were used for acquiring FAM-CGY or Rh-CGY peptidefluorescence in vivo, respectively. After the whole body mice imageswere recorded, the mice were sacrificed and the organs were dissectedand subjected to ex vivo fluorescence imaging. Data in FIG. 46 showsbrain targeting of the described invention as well as localization toorgans of the reticuloendothelial system (liver, spleen). Kidneyaccumulation may represent single chain conjugates (since they are inequilibrium with nanoparticles). Some lung accumulation also occurs (notshown).

Example 18: Sections of Brain in Peptide-Injected Mouse

Organs were fixed in a PBS solution of 4% paraformaldehyde overnight.After that, samples were placed in 15% sucrose PBS solution for 12 h,which was then replaced with 30% sucrose for 24 h. The samples were thenembedded in Tissue Tek O.C.T. compound (Mc Cormick, USA) and frozen at−60° C. in isopentane. Frozen sections, 7 mm thick, were then preparedwith a cryotome and stained with 10 μg/mL DAPI for 10 min. After PBSwashing, the sections were observed under a fluorescence microscope. Theresults in FIG. 47 show fluorescent signals in the brain tissue, withgreen coloration representing the labeled peptide and blue being DAPI.

Example 19: Imaging of Peptide Conjugate Aggregate Structures

Atomic force microscopy (AFM) analysis of FAM-CGY revealed the presenceof a heterogeneous nanoparticle and ‘nanoparticle-fibre’ network system,where some nanoparticles intercept fibres or are cross-linked by thefibres (FIG. 48a ). Nanoparticle may be perceived as nucleation siteswhere several ‘nanofiber-like’ structures of variable dimensions andheight emerge. Due to the heterogeneous nature of the network,nanoparticle tracking analysis (NTA) was employed for possible sizedistribution determination and counting of the observed species in thenanoparticle and ‘nanoparticle-fiber’ network system.

Transmission electron microscopy (TEM) investigation of the‘nanoparticle-fibre’ network showed that the ‘spherical-like’ structuresare of ‘core-shell’ morphology (FIG. 48c-g ). The core component (FIG.4a 1) is most likely an assembly of condensed unimers, dimers and/oroligomers of different arrangements (e.g., dimers with anti-parallelarrangements) and amenable to further growth through multipleinteractive forces, which eventually forms elongated ‘hair-like’projections of the shell component (FIGS. 4a 3, 4 b 1 and 4 b 2).Different nanofiber network morphologies, predominantly of twistedelongated architectures emanating from the shell component of the‘core-shell’ structures, are visible (FIG. 48c, e-g ).

The invention claimed is:
 1. A method for cellular delivery of atherapeutic from an aggregate, comprising administering a pharmaceuticalcomposition which comprises a pharmaceutically acceptable carrier andaggregate formed from a peptide-conjugate wherein the peptide-conjugatecomprises: a peptide that is a substrate for a transferrin receptor(TFRC) or a receptor for advanced glycation end product (RAGE) and thatis the peptide of SEQ ID NO. 1, the peptide of SEQ ID NO. 2 or a peptidewhich is at least 80% identical to the peptide of SEQ ID NOs. 1 or 2; acysteine residue at the N-terminus of the peptide wherein a thiol groupfrom the cysteine residue forms a disulfide bridge with otherpeptide-conjugates to promote self-assembly of the aggregate; at leastone hydrophobic molecule covalently attached to the amino group of thecysteine residue that promotes formation of the aggregate, wherein thehydrophobic molecule is selected from a compound that has at least onecarbocyclic or heterocyclic ring or a linear carbon chain of at leastthree carbon atoms, or a photoactive molecule or a fluororophore thatoperates at either visible or near-infrared spectrum; an optional linkerlocated between the at least one hydrophobic molecule and the peptide;and a therapeutic agent or pharmaceutically acceptable salt or esterthereof.
 2. The method of claim 1, wherein the aggregate is in aparticulate form having a particle size of at least 2 nm in diameter orin a fiber form having a width of at least 2 nm and a length of at least5 nm.
 3. The method of claim 1, wherein the therapeutic agent is anucleic acid comprising at least two nucleotides.
 4. The method of claim3, wherein the nucleic acid is in the form of siRNA, circular siRNA,linear DNA, plasmid DNA, shRNA, miRNA, an antisense molecule, lockednucleic acids, aptamer, peptide nucleic acids, splice modulatingoligonucleotide, nucleic acid attached to an enzyme, therapeutic nucleicacid and an expression conjugate that comprises a nucleic acid thatencodes a therapeutic protein.
 5. The method of claim 3, wherein thetherapeutic agent is covalently linked to at least one hydrophobicmolecule selected from a compound that has at least one carbocyclic orheterocyclic ring.
 6. The method of claim 5, wherein the hydrophobicmolecule linked to the therapeutic agent is a photosensitive molecule.7. The method of claim 6, where the photosensitive molecule is afluorophore operating in the visible or near infrared spectrum.
 8. Themethod of claim 7, further comprising irradiating the aggregate withelectromagnetic radiation.
 9. The method of claim 8, wherein the releaseof the therapeutic agent is accelerated following exposure to a lightsource with a broad range of electromagnetic wavelength.
 10. The methodof claim 1, wherein the therapeutic agent is physically entrapped withinthe aggregate.
 11. The method of claim 1, wherein the therapeutic agentis selected from: an active pharmaceutical grade nucleic acid, apharmaceutical grade pro-drug nucleic acid, a pharmaceutical nucleicacid conjugate and a diagnostic nucleic acid agent.
 12. The method claim1, wherein the pharmaceutical composition is in a solid form.
 13. Themethod of claim 1, wherein the pharmaceutical composition is in a liquidform.
 14. The method claim 1, further comprising contacting a mammaliancell having a transferrin receptor (TFRC) or a receptor for advancedglycation end-product (RAGE) or both receptors with the aggregate.
 15. Amethod for delivering a therapeutically active nucleic acid comprising:providing a pharmaceutically acceptable carrier and aggregate formedfrom a peptide-conjugate wherein the peptide-conjugate comprises: apeptide that is a substrate for a transferrin receptor (TFRC) or areceptor for advanced glycation end product (RAGE) and that is thepeptide of SEQ ID NO. 1, the peptide of SEQ ID NO. 2 or a peptide whichis at least 80% identical to the peptide of SEQ ID NOs. 1 or 2; acysteine residue at the N-terminus of the peptide wherein a thiol groupfrom the cysteine residue forms a disulfide bridge with otherpeptide-conjugates to promote self-assembly of the aggregate; at leastone hydrophobic molecule covalently attached to the amino group of thecysteine residue that promotes formation of the aggregate, wherein thehydrophobic molecule is selected from a compound that has at least onecarbocyclic or heterocyclic ring or a linear carbon chain of at leastthree carbon atoms, or a photoactive molecule or a fluorophore thatoperates at either visible or near-infrared spectrum; and an optionallinker located between the at least one hydrophobic molecule and thepeptide; pharmaceutically acceptable salts or esters thereof wherein theaggregate further comprises a nucleic acid; contacting a mammalian cellhaving a transferrin receptor (TFRC) or a receptor for advancedglycation end-product (RAGE) or both receptors with the aggregate; andirradiating the aggregate with a photonic source therebyphotodynamically releasing the nucleic acid.
 16. The method of claim 15,wherein the mammalian cell is from a mammalian tissue selected from thegastrointestinal tract, bone marrow, liver, spleen, brain, kidney,lungs, pancreas, bladder, eye, skin, normal and pathologic bloodvessels, and cancer cells.
 17. The method of claim 15, wherein thecontacting step further comprises administering an effective amount ofthe aggregate to a patient in need thereof to treat a disorder.
 18. Themethod of claim 17, wherein the disorder is a central nervous systemdisorder, cancer, skin cancer, bladder cancer, cardiovascular disorder,atherosclerosis, eye disorder, or diabetes.
 19. The method of claim 1,wherein the aggregate is either in the form of a spherical particle thatassociates with TFRC, or in the form of a fiber that associates withRAGE.
 20. The method of claim 15, wherein the nucleic acid is in theform of siRNA, circular siRNA, linear DNA, plasmid DNA, shRNA, miRNA, anantisense molecule, locked nucleic acids, aptamer, peptide nucleicacids, splice modulating oligonucleotide, nucleic acid attached to anenzyme, therapeutic nucleic acid and an expression conjugate thatcomprises a nucleic acid that encodes a therapeutic protein.